Bone is a highly specialized connective tissue with unique mechanical properties derived from its extensive matrix structure. A network of fibrous bundles composed of the protein collagen is presumed to provide the tension-resistant behavior of bone. In addition other materials including proteoglycans, noncollageous protein, lipids and acidic proteins associated with a mineral phase consisting primarily of poorly crystallized hydroxyapatite are deposited in the extensive matrix architecture of bone. Bone tissue is continuously renewed throughout the life of mammals. This physiologic process might serve to maintain the properties of a young tissue.
The processes of bone formation and renewal are carried out by specialized cells. Osteogenesis vis-a-vis morphogenesis and growth of bone is presumably carried out by the "osteoblasts" (bone-forming cells). Remodeling of bone is apparently brought about by an interplay between the activities of the bone-resorbing cells called "osteoclasts" and the bone-forming osteoblasts. The bony skeleton is thus not only an architectural structure with a mechanical function but also is a living tissue capable of growth, modeling, remodeling and repair. Since these processes are carried out by specialized living cells, chemical (pharmaceutical/hormonal), physical and physicochemical alterations can affect the quality, quantity and shaping of bone tissue.
A variety of pathological disorders as well as physical stress (for example, fracture) necessitate active formation of bone tissue at rates that are significantly higher than that which can be supported by the normal milieu of the body. It is thus of value to identify physiologically acceptable chemical agents (hormones/pharmaceuticals/growth factors) that can induce the formation of bone at a predetermined site. Such agents could either provide a permissive matrix structure for the deposition of bone-forming cells or cause growth stimulation of bone-forming cells or induce the differentiation of appropriate progenitors of bone-forming cells.
The presence of proteinaceous and prostaglandin-like growth stimulators for osteoblasts has been examined, see Raisz, L. G., et al., The New England Journal of Medicine, Vol. 309, No. 1, pp. 29-35 (1983) and Raisz, L. G., et al., The New England Journal of Medicine, Vol. 309, No. 2, pp. 83-89 (1983).
Urist et al. have been able to provide evidence that bone matris-associated noncollagenous proteins can be isolated by dissociative treatment of demineralized bone powder and that this mixture of extracted materials as well as partially fractionated materials obtained therefrom contain bone morphogenetic activity, see Urist, M. R., et al., Proc. Natl. Acad. Sci. USA, Vol. 76, No. 4, pp. 1828-1832 (1979); Urist, M. R., et al., Proceedings of The Society of Experimental Biology and Medicine, Vol. 162, pp. 48-53 (1979); Hanamura, H., et al., Clinical Orthopedics, Vol. 148, pp. 281-290 (1980); Urist, M. R., U. S. Pat. No. 4,294,753 (1981); Urist, M. R., et al., Clinical Orthopedics, Vol. 162, pp. 219-232 (1982); and Urist, M. R., et al., Science, Vol. 220, pp. 680-686 (1983).
Baylink and his collaborators have been able to identify a separate type of activity which presumably couples bone resorption with new bone formation, see Howard, G. A., et al., Metabolic Bone Disease and Related Research, Vol. 2, pp. 131-135 (1980); Farley, J. R., et al., Biochemistry, Vol. 21, pp. 3502-3507 (1982) and Farley, J. R., et al., Vol. 21, pp. 3508-3513 (1982). The activity which Farley et al. obtained from bone matrix involves a different extraction procedure than that of the present invention or that of Urist, supra, it has a larger molecular weight and was called "skeletal growth factor" or "skeletal coupling factor".
The procedure and techniques known in the art for obtaining putative osteogenic activities suffer from several flaws. The isolation procedures are prolonged, ill-defined and incomplete. As such, a definitive association of the activity with a chemically characterized, highly purified protein preparation has not been established. A protein of approximately 17,000 daltons obtained from calf bone powder has been termed "bone morphogenetic protein", see Urist, M. R., et al., Science, Vol. 220, pp. 680-686 (1983); it is claimed to induce efficient bone formation especially when present in a multimolecular assembly with certain other bone-derived proteins which in the absence of the 17,000 dalton protein are non-osteogenic. Urist et al., Proceedings of The Society of Experimental Biology and Medicine, Vol. 173, pp. 194-199 (1983), also identified a 17,000 to 18,000 dalton protein from human bone; this protein is claimed to induce efficient bone formation when administered together with 24,000 and 14,000 dalton human bone-derived proteins. The 24,000 and 14,000 dalton proteins are osteogenically inactive when used without the 17,000 to 18,000 dalton protein but might serve as carriers of the active 17,000 to 18,000 dalton human bone protein. Less pure preparations containing proteins of molecular weights between 17,000 and 23,000 daltons and claimed to possess bone-morphogenetic activities have been isolated from sources such as mouse osteosarcoma, see Hanamura, H., et al., Clinical Orthopedics, Vol. 153, pp. 232-240 (1980), rabbit dentin, see Conover, M. A., and Urist, M. R., The Chemistry and Biology of Mineralized Connective Tissues, Elsevier North Holland, Inc., pp. 597-606 (1981). Protein preparations used in most of the osteogenic activity measurement experimentations described in the literature to date have been of insufficient purity and thus have not led to the identification of the specific molecular entities responsible for the observed activities. Furthermore, studies reported to date have failed to reveal any chemical (biochemical) relationship between the active protein species present in the various "bone morphogenetic protein" preparations.
This invention involves the isolation, purification to an essentially homogeneous state, and the chemical description of a bone matrix protein, characterized by a novel chemical composition and amino acid sequence. This protein induces bone formation at a predetermined site when it is applied either alone or in admixture with a suitable pharmaceutically acceptable carrier. Based on the ability of this essentially homogeneous protein to induce bone development, it is sometimes referred to herein as the "primary osteogenic factor" of bone. Two additional bone-derived protein preparations unrelated to the previously described protein are also described; these proteins by themselves do not induce bone formation but may positively influence osteogenesis when used inconjunction with the primary osteogenic factor.
The invention also concerns the isolation and characterization of immunologically related osteogenic proteins from various bone sources. A rapid, precise and efficient isolation technique is described for the purification of the members of an immunologically related family of equivalent proteins related to each of the above-noted protein species obtained from bone.